The recognition of the hierarchies of genetic pathways coordinating heart development remains incomplete, in part because of the difficulties to identify the critical cis-acting sequences that are required for the regionalized expression of genes during cardiogenesis. Here we propose an integrative system to identify the critical components of the regulatory network of TBX20, a gene that plays important roles in heart development. To achieve this, we propose to utilize a combination of an in vivo zebrafish and mouse reporter assay to identify heart enhancers in the TBX20 locus. We will carry out a saturation scan for enhancers in the TBX20 locus, systematically testing sequences regardless of their pattern of evolutionary conservation, with the goal of identifying the critical enhancers that coordinate TBX20 expression during cardiogenesis. We will carry out a detailed characterization of the spatial and temporal domains of these heart enhancers during embryogenesis, leading to a broad understanding of the components of the TBX20 regulatory network. We will test the necessity of each of these enhancers for the proper expression of TBX20. Using engineered Bacterial Artificial Chromosomes (BACs), we will delete each enhancer that drives expression in multiple domains of the heart. This experimental design will allow us to infer the necessity of individual components of the regulatory network for the proper temporal, spatial and quantitative expression of TBX20. In summary, we will use zebrafish and mouse in vivo assays to characterize the cis-regulatory network that coordinates TBX20 expression during cardiogenesis, and evaluate how the individual components of the network orchestrate the dynamic and complex pattern of TBX20 expression.